Paclitaxel (also can be called TAXOL®), isolated from Taxus brevifolia[1], was found to show anti-tumor activity by US National Cancer Institute (NCI). Premier mechanistic study indicated that paclitaxel is a mitotic inhibitor, which arrest the growth of cancer cells at G2 and M stage by promoting polymerization and depolymerization of cancer cell microtubule, then preventing formation of spindle in cancer cell[2]. Further mechanistic study indicated paclitaxel can also be used as bacterium lipopolysaccharide (LPS) analogue, which exerts its anti-tumor effect by affecting or changing the function of macrophages in immune system, for example, by inducing the expression of tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) in maerophages[3, 4]. Furthermore, it shows anti-tumor effect by activating MAP-2 kinase, and/or promoting tyrosine phosphorylation of cancer cells[5, 6].
Muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP) is the minimal structural unit shows immunoadjuvant activity among mycobacterium cell wall peptidoglycans[7, 8]. MDP, injected at the same time with or before the injection of antigen, will enhance immune response or change immune response type. Furthermore, Muramyl dipeptide shows other activities, such as nonspecific resistance to infection caused by, for example, pneumobacillus, colibacillus, pseudomonas aeruginosa, mononucleosis listeria, and/or tritirachium album etc, nonspecific resistance to, for example, fibrosarcoma and hepatoma etc, and immunoregulation[9-13]. Studies also indicated that MDP together with lipopolysaccharide (LPS) can significantly stimulates the cytokines expression of macrophage[14-16].
Based on these, we expected that paclitaxel together with muramyl dipeptide may show synergistic effect as well. We are the first to propose the new idea that bonding chemotherapy drug paclitaxel and immunostimulants muramyl dipeptide to form a series of conjugates. Biological tests are carried out to prove effectiveness of the new idea, which—combines chemotherapy and immunotherapy to realize anti-tumor and anti-metastatic effects[17].
Applicants disclosed two types of conjugates in our previous patent application[18], which were obtained by bonding muramyl dipeptide with paclitaxel 2′-hydroxy (2′-O-MTC, Structure 1), or with 3′-amino of 3′-N benzoyl paclitaxel (3′-N-MTC, Structure 1). In in vitro tests, Applicants found that 2′-O-MTC conjugate not only maintained anticancer activity of paclitaxel, but also assisted macrophages to produce αTNF- and IL-1 significantly, which means it potentially can inhibit metastasis. However, the activity of 3′-N-MTC conjugate was not significant. Based on that, we preliminarily determined the optimal position of conjugates for bonding would be paclitaxel's 2′-hydroxyl group. Unfortunately, 2′-O-MTC conjugate did not show desired results in vivo, which might depend on the physicochemical properties or the pharmaceutical properties of the molecule. To continue this design concept used in the new drug discovery, Applicants optimized the 2′-O-MTC conjugate by simplifing structures of muramyl dipeptide molecules, and obtained a new series of 2′-O-MTC analogues showing significant anti-tumor and anti-metastasis activities in vivo, which means they can be developed as antitumor drugs. Disclosed herein are the aforementioned new series of 2′-O-MTC analogues.

Paclitaxel is a taxanes antineoplastic drug, while docetaxel (Structure 2), a semisynthetic derivative of Paclitaxel, is another important member of taxanes antineoplastic drug which shows inhibitory activities against terminal breast cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, liver cancer, head and neck tumors. Current research indicated that docetaxel induces the apoptosis of cancer cell by promoting microtubule to form stable polymer, inhibiting depolymerization[19], and furthermore inhibiting mitosis and proliferation of cancer cell[20]. Research also discovered that docetaxel can make the tumor cell stop at G2/M stage by up regulating Bax protein expression and down regulating Bcl-2 protein expression[21]. Based on this, the disclosure of this application involves replacing paclitaxel in the original conjugates with docetaxel to form conjugates of docetaxel-muramyl dipeptides (MDC), which also showed anti-tumor activities.

Muramyl dipeptide shows broad biological activities, and attracts great interest when discovered. However, muramyl dipeptide shows several side effects, such as immunogen induced allergic reactions, fever, inflammation and sleepiness, which limit its clinical application. In order to find muramyl dipeptide analog with higher activity and fewer side effects, scientists have synthesized hundreds of muramyl dipeptide simplifiers or analogues, and studied their biological activities. L-threonine-Muramyl dipeptide is obtained by replacing L-alanine in muramyl dipeptide molecule with L-threonine, which shows higher immunoadjuvant activity than that of the muramyl dipeptide, but pyrogen is 100 times lower. When used as a vaccine adjuvant, L-threonine-Muramyl dipeptide doesn't stimulate macrophages and anti-inflammatory effects, but stimulates the immune response of the administered antigen, so it can be an ideal vaccine adjuvant because its activity and side effect can be effectively separated[22].
Murabutide is obtained by introducing muramyl dipeptide to long lipotropic chain. Murabutide can enhance non-specific anti-bacterial and anti-viral infection of host immune system, and induce activity of colony stimulating factor, Also, it is well tolerated by human[23-26]. Compared to other exogenous immunomodulators, Murabutide is non-pyrogenicity and promotes cytokines, both synergetically and selectively, to release Th1 cytokine, and Murabutide does not cause inflammatory response[27, 28]. Furthermore, Murabutide combined with IFN-α or IL-2 can significantly enhance the anti-tumor activities of the cytokines, hence improve the anti-viral and anti-inflammatory effect of IFN-α[29, 30]. Murabutide can regulate function of macrophage[31]. It can also be used in the treatment of chronic hepatitis C (HCV), because of the synergistic effect shown in vitro when combined with IFN-α[32].
Muramyl tripeptidephosphatidylethanolamine (MTP-PE) is obtained by introducing lipophiliclong chain to muramyl dipeptides through phosphate bond. MTP-PE can activate monocytes and macrophages, then kill tumor cells. MTP-PE encapsulated in liposomes (L-MTP-PE), injected intravenously, is mainly directed to activate the macrophages in lung, liver and spleen[33], wherein its activities is increased by ten to hundreds times, and pyrogenicity is significantly reduced. Two hours after being intravenously injected to metastatic melanoma patients, tumor necrosis factor in plasma increased in sixteen times, and the level of neopterin and interleukin was effectively improved[34].
MDP-Lys (L18) is obtained by introducing lipophilic long chain to muramyl dipeptides through lysine. MDP-Lys (L18) can enhance the production of cytokines such as CSFs, IL-1, IL-6, tumor necrosis factor (TNF-α) etc, which play important role in regulation of the hematopoietic system[35, 36]. In addition, MDP-Lys (L8) has a strong anti-infection, anti-tumor activity[37].
MDP-C is obtained by introducing aromatic conjugate system to muramyl dipeptides through lysine. MDP-C can induce macrophage to generate cytotoxic activity against P388 leukemia cells, it can also induce Tlymphocytes (CTLs) to generate cytotoxic activity against mastocytoma P815. It is reported that the MDP-C stimulates mouse bone marrow dendritic cells (BMDCs) to produce cytokines IL-2 and IL-12 (interleukin), and it also can be used as effective immunopotentiator for it shows activity on stimulating cytotoxic Tlymphocytes to produce interferon-γ. Low doses of MDP-C can significantly and synergistically promote proliferation of mouse spleen lymphocyte induced by Concanavalin A (ConA). In addition, MDP-C can increase the expression of bone marrow dendritic cell surface molecules, such as CD11c, MHC land cell adhesion molecule-1. Also, MDP-C, in vitro, can significantly enhance, through producing antibodies and specific hepatitis B virus surface antigen (HBsAg) Tcell response, the response of immune system to the HBsAg in hepatitis B virus transgenic mice[38, 39].
Adamantantylamide dipeptide (AdDP) is obtained by bonding carboxyl teminal of dipeptide fragment in muramyl dipeptide molecule with amantadine. AdDP is safe, and shows anti-virus infection activity. Compared with other MDP analogues, its bioavailability is higher[40]. AdDP can enhance the humoral immunity both in BALB/c mice and rabbit when administered with protein immunogen orally or peritoneally[41].
Chemists also obtained muramyl dipeptide sugar-free ring analogs by synthesis or isolating from natural product, such as FK-156 and FK-565. They show anti-infection, anti-viral and anti-tumor activities[42].